Gene transfer into hematopoietic stem cells (HSCs) has the potential to treat many diseases, including hemoglobinopathies, immunodeficiencies, infectious diseases, and even disorders of other organ systems if HSCs form different tissue types. However the oncogenic risks of insertional mutagenesis and low transduction rates of human HSCs remain major problems. Here we propose to develop retroviral vectors based on foamy virus (FV) in order to overcome these problems. Wild-type FV is a non-pathogenic virus common in captive primates that has never been linked to oncogenesis, suggesting that FV vectors may be safer than other integrating vectors. In order to establish their oncogenic risk, we will study insertional mutagenesis by FV vectors in a variety of ways, including mapping of integration sites in human CD34+ cells; correlating provirus sites with chromosomal gene location, expression, and conformation; and determining if and how neighboring genes are activated in a panel of cells with mapped proviruses. Several assays will be developed to look at a vector s potential for activating neighboring genes, including plasmid transfection assays with cloned proviruses; celt transformation assays based on provirus-dependent activation of cytokine independence; and bone marrow transplantation assays in cancer-prone mice to directly demonstrate oncogenesis. These assays will be used to compare different FV vectors and also vectors based on lentiviruses and murine leukemia viruses. Safer FV Vectors will be designed that have less potential for activating neighboring genes by incorporating internal promoters with less enhancer activity, changing vector backbone elements to prevent read-through transcription and splicing, and including genetic insulator elements. In prior work we showed that FV vectors efficiently transform mouse and human HSCs in animal transplantation and xenotransplantation models, suggesting that they may be superior to other types of stem cell vectors. Here we will determine HSC transduction rates in large animal models that more closely simulate human hematopoiesis (dog and non-human primate). FV vectors will be compared to tentivirus and murine leukemia virus vectors in these transplantation studies to determine which vector type is more effective, and to compare potential side effects. These studies will establish useful methods for assessing the risks of any integrating vector, improve existing FV vectors, and direcdy compare the safety and efficacy of all 3 types of retroviral vectors.